Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.